The present invention relates to a method of assessing a diabetic patient's risk of experiencing a diabetes-associated pathologic condition, or the efficacy of therapeutic intervention in prevention of diabetic complications, by measuring the levels of certain, newly discovered, phosphorylated carbohydrates present in the cellular components of biological materials, such as red blood cells. Some of these metabolites are unstable, and decompose to produce reactive, toxic materials, such as 3-deoxyglucosone produced from fructose-3-phosphate. As will appear below, these metabolites have a demonstrable toxic effect on red blood cells. In addition, compounds such as 3-deoxyglucosone, due to their uncharged character, are able to diffuse out of the red blood cells into the patient's biological fluids such as blood plasma, lymph fluids and interstitial fluids where they then react with proteins. Such reactions, known as non-enzymatic glycosylation or Maillard reactions, lead to the formation of advanced glycosylation end products (AGE) and crosslinking of long lived proteins such as collagen and basement membrane components.
The reaction of reducing sugars with proteins to form stable adducts is relatively well established. In 1912, Maillard experimentally identified the reaction responsible for the formation of the brown pigments often observed during the cooking of food. By heating glucose, as well as certain other saccharides, with various amino acids, Maillard was able to form stable brown pigments. L. C. Maillard C. R. Acad. Sci., 154:66 (1912).
The mechanism of this non-enzymatic glycosylation was elucidated by food chemists in subsequent years; because the resultant browning changes the flavor and nutritional value of foods, especially during storage and processing (e.g., heating), the food industry quickly recognized the economic importance of this reaction and the need to understand and manipulate it. Further insights concerning the reaction were gained after the more recent discovery that the Maillard reaction occurs in vivo, including in humans, and is implicated in aging and diabetes-associated pathologic conditions.
The Maillard reaction, as currently understood, may be divided into early and advanced stages. It is initiated by the reversible non-enzymatic condensation of a reducing sugar with a free amino group of a protein, nucleic acid, or amine to form a Schiff base. The Schiff base reversibly undergoes an acid catalyzed Amadori rearrangement to give a stable ketamine compound called the Amadori product (also called the early glycosylation or early Maillard end-product). This early stage of the reaction is often termed non-enzymatic glycosylation or glycation. Although coined to describe the reaction wherein glucose is the sugar substrate, here and throughout this application the terms "non-enzymatic glycosylation", "non-enzymatic glycation" and similar terms are used interchangeably to refer to the early stages of the Maillard reaction, regardless of whether the sugar substrate is glucose or some other reactive reducing sugar (e.g., fructose). In the advanced stage, the Amadori product irreversibly breaks down to a variety of reactive .alpha.-dicarbonyl compounds, also known as breakdown products, the relative amounts of which are determined by reaction conditions. These reactive dicarbonyls, which include the deoxyglucosones, are generally more reactive than the parent sugar toward free amino groups. These compounds further react with free amino groups to form various stable, UV absorbent, often fluorescent, cross-linked products (also called advanced glycosylation or advanced Maillard end-products). In some cases, the reaction appears to terminate at this point, but often, these end-products further polymerize to give the characteristic brown pigments. See generally, The Maillard Reaction in Aging, Diabetes and Nutrition, J. W. Baynes and V. M. Monnier, eds. (Liss, New York, 1989).
The similarities between the pathologies arising from aging and those resulting from diabetes have been extensively reported. Studies have shown that many diabetes-associated pathologic conditions are clinically very similar to the pathologies normally associated with aging. Here and throughout this application the term "diabetes-associated pathologic condition" and synonymous terms are meant to include the various well-known neuropathic, nephropathic, macroangiopathic as well as other complications of diabetes. These conditions include, but are not limited to, the following representative examples. In cataracts are found to occur 10-15 years earlier than in normal individuals. Arteries and joints prematurely stiffen, lung elasticity and vital capacity prematurely decrease, and atherosclerosis, myocardial infarction and strokes occur more frequently in diabetics than in age-matched nondiabetic individuals. Diabetics are more susceptible to infection, and are more likely to have hypertension, accelerated bone loss, osteoarthritis and impaired T-cell function at a younger age than nondiabetics. Id., at p. 5 and references cited therein.
The similarities between aging and diabetes-associated pathologic conditions would appear to suggest a common mechanistic rationale. A variety of mechanisms have been proposed as a common biochemical basis for both diabetes-associated pathologic conditions and aging, however, none of the proposed mechanisms has proven especially satisfactory. The hypothesis most strongly supported by data from human subjects is premised on a non-enzymatic glycosylation mechanism. This hypothesis states that the aging process and diabetes-associated pathologic conditions, such as those described above are caused, at least in part, by protein modification and cross-linking by glucose and glucose-derived metabolites via the Maillard reaction. V. M. Monnier, R. R. Kohn, A. Cerami, Proc. Natl. Acad. Sci. U.S.A., 81:583 (1984); J. H. Lee, D. H. Shin, A. Lupovitch, D. X. Shi, Biochem. Biophys. Res. Commun., 123:888 (1984). In the case of diabetic complications, the reaction is thought to be kinetically accelerated by the chronic hyperglycemia often associated with diabetes. Evidence supporting this mechanism includes the fact that long-lived proteins such as collagen and lens crystallins from diabetic subjects are significantly more glycosylated, and therefore, modified and cross-linked, than those from age-matched normal controls. Thus, the unusual incidence of cataracts in diabetics at a relatively early age is explainable, according to the glycosylation hypothesis, by the increased rate of modification and cross-linking of lens crystalline, which is driven by hyperglycemia. Similarly, the early onset of joint and arterial stiffening as well as loss of lung capacity observed in diabetics may be explained by the increased rate of modification and cross-linking of collagen, the key structural protein. Because these proteins are long-lived, the consequences of glycosylation tend to be cumulative, and thus more drastic than in proteins with a relatively high turnover.
Methods for monitoring metabolic control in diabetic patients by measurement of glycosylation end-products are known. The concentration of glycosylated hemoglobin is known to reflect mean blood glucose concentration during the preceding several weeks. U.S. Pat. No. 4,371,374, issued to A. Cerami et al., described a method for monitoring glucose levels by quantitation of the degradation products of glycosylated proteins, more specifically non-enzymatically glycosylated amino acids and peptides, in urine. The described method utilized the affinity of alkaline boronic acids for forming specific complexes with the coplanar cis-diol groups found in glycosylation end-products to separate and quantitate such end-products.
U.S. Pat. No. 4,761,368, issued to A. Cerami, describes the isolation and purification of a chromophore present in browned polypeptides, e.g., bovine serum albumin and poly-L-lysine. The chromophore, 2-(2-furoyl)-4(5)-2(furoyl)-1H-imidazole (FFI) is a conjugated heterocycle derived from the condensation of two molecules of glucose with two lysine-derived amino groups. The '368 patent further describes the use of FFI in a method for measuring "aging" (the degree of advanced glycosylation) in a protein sample wherein the sample "age" is determined by measuring the amount of the above-described chromophore in the sample and then comparing this measurement to a standard (a protein sample having an amount of FFI which has been correlated to the "age" of the sample).
A difficult problem encountered in the medical treatment of diabetes has been the inability to identify those diabetic patients who are at risk of experiencing some diabetes-associated pathologic condition, so as to effect timely intervention. However, recent reports have identified certain diabetes-associated metabolites in mammalian lens tissue previously undetected in vivo, namely, sorbitol-3-phosphate (S3P) (Szwergold, B. S., Kappler, F., Brown, T. R., Pfeffer, P., and Osman, S. F., J. Biol. Chem., 264:9278 (1989) and fructose-3-phosphate (F3P) (Szwergold, B. S., Kappler, F., and Brown, T. R., Science, 247:451 (1990). The concentrations of both of these compounds in the lens of the diabetic rat have been found to increase substantially after onset of the disease. Gonzalez, R. G., Mag. Res. Med., 6:435 (1988). Our research has shown that F3P is a relatively potent glycosylating agent which, in addition, is quite labile, especially in the presence of amines, breaking down to produce an even more potent Maillard glycosylating and cross-linking agent, 3-deoxyglucosone (3dG). Kato, M., Hayase, F., Shin, D. B., Oimomi, M. and Baba, S. (1989), The Maillard Reaction in Aging, Diabetes and Nutrition, J. W. Baynes and V. M. Monnier, eds, at 69-84 (Liss, New York, 1989).
Since, as described above, increased glycosylation of proteins in diabetic patients is well known and appears to be casually related to various pathologic conditions, the increase in F3P concentration in diabetic rat lenses suggests that F3P and 3dG may be casually linked to the observed enhanced glycosylation.